Substituted pyrimidine derivatives

ABSTRACT

This invention relates to compounds of Formula I, 
                         
a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a prodrug thereof, or a pharmaceutically acceptable salt of a prodrug thereof. The compounds interact with CRF 1  receptors, including human CRF 1  receptors. This invention also relates to methods of using the compounds of the invention to treat a disorder or condition, the treatment of which can be effected or facilitated by antagonizing a CRF receptor, such as CNS disorders or diseases, particularly anxiety-related disorders such as anxiety, and mood disorders such as major depression.

FIELD OF THE INVENTION

The present invention relates generally to compounds that bind to CRFreceptors, and particularly to substituted pyrimidine derivativesderivatives as CRF₁ receptor antagonists and to the use thereof as atreatment for disorders that are associated with CRF or CRF₁ receptors.

BACKGROUND OF THE INVENTION

Corticotropin releasing factor (CRF) is a 41 amino acid peptide that isthe primary physiological regulator of proopiomelanocortin (POMC)derived peptide secretion from the anterior pituitary gland [J. Rivieret al., Proc. Natl. Acad. Sci (USA) 80:4851 (1983); W. Vale et al.,Science 213:1394 (1981)]. In addition to its endocrine role at thepituitary gland, CRF is known to have a broad extrahypothalmicdistribution in the CNS, contributing therein to a wide spectrum ofautonomic behavioral and physiological effects consistent with aneurotransmitter or neuromodulator role in the brain [W. Vale et al.,Rec. Prog. Horm. Res. 39:245 (1983); G. F. Koob, Persp. Behav. Med. 2:39(1985); E. B. De Souza et al., J. Neurosci. 5:3189 (1985)]. There isevidence that CRF plays a significant role in integrating the responsein the immune system to physiological, psychological, and immunologicalstressors, in psychiatric disorders and neurological diseases includingdepression, anxiety-related disorders and feeding disorders, and in theetiology and pathophysiology of Alzheimer's disease, Parkinson'sdisease, Huntington's disease, progressive supranuclear palsy andamyotrophic lateral sclerosis, as they relate to the dysfunction of CRFneurons in the central nervous system [J. E. Blalock, PhysiologicalReviews 69:1 (1989); J. E. Morley, Life Sci. 41:527 (1987); E. B. DeSouze, Hosp. Practice 23:59 (1988)].

There is evidence that CRF plays a role in mood disorders. Mooddisorders, also known as affective disorders, are well recognized in theart and include depression, including major depression, single episodedepression, recurrent depression, child abuse induced depression, andpostpartum depression; dysthemia; bipolar disorders; and cyclothymia. Itwas shown that in individuals afflicted with affective disorder, ormajor depression, the concentration of CRF in the cerebral spinal fluid(CSF) is significantly increased. [C. B. Nemeroff et al., Science226:1342 (1984); C. M. Banki et al., Am. J. Psychiatry 144:873 (1987);R. D. France et al., Biol. Psychiatry 28:86 (1988); M. Arato et al.,Biol. Psychiatry 25:355 (1989)]. Furthermore, the density of CRFreceptors is significantly decreased in the frontal cortex of suicidevictims, consistent with a hypersecretion of CRF [C. B. Memeroff et al.,Arch. Gen. Psychiatry 45:577 (1988)]. In addition, there is a bluntedadrenocorticotropin (ACTH) response to CRF (i.v. administered) observedin depressed patients [P. W. Gold et al., Am. J. Psychiatry 141:619(1984); F. Holsboer et al., Psychoneuroendocrinology 9:147 (1984); P. W.Gold et al., New Engl. J. Med. 314:1129 (1986)]. Preclinical studies inrats and non-human primates provide additional support for thehypothesis that hypersecretion of CRF may be involved in the symptomsseen in human depression [R. M. Sapolsky, Arch. Gen. Psychiatry 46:1047(1989)]. There is also preliminary evidence that tricyclicantidepressants can alter CRF levels and thus modulate the numbers ofreceptors in the brain [Grigoriadis et al., Neuropsychopharmacology 2:53(1989)].

CRF has also been implicated in the etiology of anxiety-relateddisorders. Anxiety disorders are a group of diseases, recognized in theart, that includes phobic disorders, anxiety states, post-traumaticstress disorder, generalized anxiety disorder, social anxiety disorder,anxiety with co-morbid depressive illness, panic disorder,obsessive-compulsive disorder, and atypical anxiety disorders [The MerckManual of Diagnosis and Therapy, 16^(th) edition (1992)]. Emotionalstress is often a precipitating factor in anxiety disorders, and suchdisorders generally respond to medications that lower response tostress. Excessive levels of CRF are known to produce anxiogenic effectsin animal models [see, e.g., Britton et al., 1982; Berridge and Dunn,1986 and 1987]. Interactions between benzodiazepine/non-benzodiazepineanxiolytics and CRF have been demonstrated in a variety of behavioralanxiety models [D. R. Britton et al., Life Sci. 31:363 (1982); C. W.Berridge and A. J. Dunn, Regul. Peptides 16:83 (1986)]. Studies usingthe putative CRF receptor antagonist α-helical ovine CRF (9–41) in avariety of behavioral paradigms demonstrates that the antagonistproduces “anxiolytic-like” effects that are qualitatively similar to thebenzodiazepines [C. W. Berridge and A. J. Dunn, Horm. Behav. 21:393(1987), Brain Research Reviews 15:71 (1990); G. F. Koob and K. T.Britton, In: Corticotropin-Releasing Factor: Basic and Clinical Studiesof a Neuropeptide, E. B. De Souza and C. B. Nemeroff eds., CRC Press p.221 (1990)]. Neurochemical, endocrine and receptor binding studies haveall demonstrated interactions between CRF and benzodiazepineanxiolytics, providing further evidence for the involvement of CRF inthese disorders. Chlordiazepoxide attenuates the “anxiogenic” effects ofCRF both in the conflict test [K. T. Britton et al., Psychopharmacology86:170 (1985); K. T. Britton et al., Psychopharmacology 94:306 (1988)]and in the acoustic startle test [N. R. Swerdlow et al.,Psychopharmacology 88:147 (1986)] in rats. The benzodiazepine receptorantagonist Ro 15-1788, which was without behavioral activity alone inthe operant conflict test, reversed the effects of CRF in adose-dependent manner while the benzodiazepine inverse agonist FG 7142enhanced the actions of CRF [K. T. Britton et al., Psychopharmacology94:396 (1988)].

Use of CRF₁ antagonists to treat of Syndrome X has also been describedin EP 1097709A2.

Use of CRF₁ antagonists to treat congestive heart failure is describedin U.S. Pat. No. 6,043,260.

It has also been suggested that CRF₁ antagonists are useful for treatingarthritis and inflammation disorders [E. L. Webster et al., J. Rheumatol29:1252 (2002); E. P. Murphy et al., Arthritis Rheum 44:782 (2001)];stress-related gastrointestinal disorders [K. E. Gabry et al., MolecularPsychiatry 7:474 (2002)]; and skin disorders [C. C. Zouboulis et al.,Proc. Natl. Acad. Sci. 99:7148 (2002)].

It was disclosed recently that, in an animal model, stress-inducedexacerbation of chronic contact dermatitis is blocked by a selectiveCRF₁ antagonist, suggesting that CRF₁ is involved in the stress-inducedexacerbation of chronic contact dermatitis and that CRF₁ antagonist maybe useful for treating this disorder [K. Kaneko et al., Exp Dermatol,12:47 (2003)].

WO 01/60806 discloses aryl piperazines compounds that can bind with highaffinity and high selectivity to CRF₁ receptors.

It is an object of the invention to provide novel substituted pyrimidinederivative compounds.

It is another object of the invention to provide novel substitutedpyrimidine derivatives that are useful as CRF₁ receptor antagonists.

It is another object of the invention to provide novel substitutedpyrimidine compounds as treatment of disorders or conditions that areassociated with CRF or CRF₁ receptors, such as anxiety disorders,depression, and stress related disorders.

It is another object of the invention to provide a method of treatingdisorders or conditions that are associated with CRF or CRF₁ receptors,such as anxiety-related disorders, mood disorders, and stress relateddisorders.

It is yet another object of the invention to provide a pharmaceuticalcomposition useful for treating disorders or conditions that areassociated with CRF or CRF₁ receptors, such as anxiety-relateddisorders, mood disorders, and stress related disorders.

There are other objects of the invention which will be evident orapparent from the description of the invention in the specification ofthe application.

SUMMARY OF THE INVENTION

The present invention provides a compound of Formula I,

a stereoisomer thereof, a pharmaceutically acceptable salt thereof, aprodrug thereof, or a pharmaceutically acceptable salt of a prodrugthereof, wherein:

X is selected from —NR₃R₄, —OR₃, —CR₃R₅R₅, —C(O)R₃, —S(O)_(m)R₃,—NR₃C(O)R₄, or —NR₃S(O)_(m)R₄;

m is 0, 1 or 2;

G is selected from N or C(R₂);

R₁ and R₂ are independently selected from —H, —NH(alkyl), —N(alkyl)₂,—NH(substituted alkyl), —N(substituted alkyl)₂, —O(alkyl),—O(substituted alkyl), halogen, alkyl, substituted alkyl, haloalkyl,cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, substituted aryl,heterocycloalkyl, substituted heterocycloalkyl, substituted heteroaryl,—CR₅R₅Ar, —OAr, —S(O)_(m)Ar, —NR₅Ar, —S(O)_(m)alkyl,—S(O)_(m)substituted alkyl, —CN, —NO₂, —OH, —NH₂, —SH, —C(O)NR₄R₅ and—C(S)NR₄R₅;

R₃ and R₄ are independently selected from heteroaryl, substitutedheteroaryl, aryl cycloalkyl, substituted aryl cycloalkyl, heteroarylcycloalkyl, substituted heteroaryl cycloalkyl, aryl heterocycloalkyl,substituted aryl heterocycloalkyl, heteroaryl heterocycloalkyl,substituted heteroaryl heterocycloalkyl, heterocycloalkyl or substitutedheterocycloalkyl, provided when both R₃ and R₄ are present one of the R₃or R₄ is selected from a group provided herein above and the other R₃ orR₄ is selected from —H, alkyl, substituted alkyl, haloalkyl, cycloalkyl,substituted cycloalkyl, aryl, heteroaryl, heterocycloalkyl, substitutedheterocycloalkyl, substituted heteroaryl, aryl cycloalkyl, substitutedaryl cycloalkyl, heteroaryl cycloalkyl, substituted heteroarylcycloalkyl, aryl heterocycloalkyl, substituted aryl heterocycloalkyl,heteroaryl heterocycloalkyl, or substituted heteroaryl heterocycloalkyl.Ar is selected from aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; and

R₅ each is independently selected from —H, alkyl, cycloalkyl, andhaloalkyl, wherein alkyl may be substituted with 1–3 substituentsselected from halogen, —O(alkyl), —NH(alkyl), —N(alkyl)₂,—C(O)NH(alkyl), —C(O)N(alkyl)₂, —NHC(O)alkyl, —N(alkyl)C(O)alkyl, and—S(O)_(m)alkyl, heterocycloalkyl, substituted heterocycloalkyl and Ar.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of Formula I, a stereoisomer thereof,a pharmaceutically acceptable salt thereof, a prodrug thereof, or apharmaceutically acceptable salt of a prodrug thereof. The compositionscan be prepared in any suitable forms such as tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols, and ointments.

The compounds of the inventions are CRF₁ receptor antagonists. Thus, inanother aspect, the present invention provides a method of antagonizingCRF₁ receptors in a warm-blooded animal, comprising administering to theanimal a compound of the invention at amount effective to antagonizeCRF₁ receptors.

In still another aspect, the present invention provides a method forscreening for ligands for CRF₁ receptors, which method comprises: a)carrying out a competitive binding assay with CRF₁ receptors, a compoundof Formula I which is labeled with a detectable label, and a candidateligand; and b) determining the ability of said candidate ligand todisplace said labeled compound.

In still another aspect, the present invention provides a method fordetecting CRF₁ receptors in a tissue comprising: a) contacting acompound of Formula I, which is labeled with a detectable label, with atissue, under conditions that permit binding of the compound to thetissue; and b) detecting the labeled compound bound to the tissue.

In yet another aspect, the present invention provides a method ofinhibiting the binding of CRF to CRF₁ receptors in vitro, comprisingcontacting a compound of the invention with a solution comprising cellsexpressing the CRF₁ receptor, such as IMR32 cells, wherein the compoundis present in the solution at a concentration sufficient to inhibit thebinding of CRF to the CRF₁ receptor.

Compounds of the invention are useful for treating, in a warm-bloodedanimal, particularly a mammal, and more particularly a human, variousdisorders that are associated with CRF or CRF₁ receptors, or disordersthe treatment of which can be effected or facilitated by antagonizingCRF₁ receptors. Examples of such disorders include anxiety-relateddisorders (such as anxiety states, generalized anxiety disorder, socialanxiety disorder, anxiety with co-morbid depressive illness, panicdisorder, and obsessive-compulsive disorder phobic disorders,post-traumatic stress disorder, and atypical anxiety disorders); mooddisorders, also known as affective disorders (such as depression,including major depression, single episode depression, recurrentdepression, child abuse induced depression, and postpartum depression;dysthemia; bipolar disorders; and cyclothymia); post-traumatic stressdisorder; supranuclear palsy; immune suppression; drug or alcoholwithdrawal symptoms; substance abuse disorder (e.g., nicotine, cocaine,ethanol, opiates, or other drugs); inflammatory disorders (such asrheumatoid arthritis and osteoarthritis); fertility problems includinginfertility; pain; asthma; psoriasis and allergies; phobias; sleepdisorders induced by stress; pain perception (such as fibromyalgia);dysthemia; bipolar disorders; cyclothymia; fatigue syndrome;stress-induced headache; cancer; human immunodeficiency virus (HIV)infections; neurodegenerative diseases (such as Alzheimer's disease,Parkinson's disease and Huntington's disease); gastrointestinal diseases(such as ulcers, irritable bowel syndrome, Crohn's disease, spasticcolon, diarrhea, and post operative ilius and colonic hypersensitivityassociated by psychopathological disturbances or stress); eatingdisorders (such as anorexia and bulimia nervosa and other feedingdisorders); hemorrhagic stress; stress-induced psychotic episodes;euthyroid sick syndrome; syndrome of inappropriate antidiarrhetichormone (ADH); obesity; head traumas; spinal cord trauma; ischemicneuronal damage (e.g., cerebral ischemia such as cerebral hippocampalischemia); excitotoxic neuronal damage; epilepsy; cardiovascular andheart related disorders (such as hypertension, tachycardia andcongestive heart failure); stroke; immune dysfunctions including stressinduced immune dysfunctions (e.g., stress induced fevers, porcine stresssyndrome, bovine shipping fever, equine paroxysmal fibrillation, anddysfunctions induced by confinement in chickens, sheering stress insheep or human-animal interaction related stress in dogs); muscularspasms; urinary incontinence; senile dementia of the Alzheimer's type;multiinfarct dementia; amyotrophic lateral sclerosis; chemicaldependencies and addictions (e.g., dependences on alcohol, cocaine,heroin, benzodiazepines, or other drugs); osteoporosis; psychosocialdwarfism, hypoglycemia, and skin disorders (such as acne, psoriasis,chronic contact dermatitis, and stress-exacerbated skin disorders). Theyare also useful for promoting smoking cessation and hair growth, ortreating hair loss.

Thus, in yet a further aspect the present invention provides a method oftreating a disorder, in warm-blooded animal, the treatment of whichdisorder can be effected or facilitated by antagonizing CRF₁ receptors,which method comprises administration to a patient in need thereof aneffective amount of a compound of Formula I. In a particular embodimentthe invention provides a method of treating disorders that manifesthypersecretion of CRF. Examples of disorders that can be treated withthe compounds of the invention include generalized anxiety disorder;social anxiety disorder; anxiety; obsessive-compulsive disorder; anxietywith co-morbid depressive illness; panic disorder; and mood disorderssuch as depression, including major depression, single episodedepression, recurrent depression, child abuse induced depression,postpartum depression, hair loss, and contact dermatitis. It ispreferred that the warm-blooded animal is a mammal, and more preferredthat the animal is a human.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of Formula I described above.

Particularly preferred compounds of general Formula I are those where Xis —NR3R4.

Other preferred compounds of the invention are those of general FormulaI where X is —NR₃R₄ and one of R₃ or R₄ is aryl cycloalkyl or heteroarylcycloalkyl.

Other preferred compounds of the invention are those of general FormulaI where X is —NR₃R₄ and one of R₃ or R₄ is aryl cycloalkyl or heteroarylcycloalkyl and the point of attachment is the cycloalkyl ring.

Other preferred compounds of the invention are those of general FormulaI where X is —NR₃R₄ and one of R₃ or R₄ is aryl cycloalkyl or heteroarylcycloalkyl and the point of attachment is the cycloalkyl ring and one ofR₃ or R₄ is hydrogen.

Other preferred compounds of the invention are those of general FormulaI where X is —NR₃R₄ and one of R₃ or R₄ is substituted aryl cycloalkylor substituted heteroaryl cycloalkyl and the point of attachment is thecycloalkyl ring and one of R3 or R4 is hydrogen.

Other preferred compounds of the invention are those of general FormulaI where X is —NR₃R₄ and one of R₃ or R₄ is substituted aryl cycloalkylor substituted heteroaryl cycloalkyl where the substituent is eitheralkyl or alkoxy and is on the cycloalkyl ring and the point ofattachment is the cycloalkyl ring and one of R₃ or R₄ is hydrogen.

Other preferred compounds of the invention are those of general FormulaI where X is —NR₃R₄ and one of R₃ or R₄ is substituted aryl cycloalkylor substituted heteroaryl cycloalkyl where the substituent is eitheralkyl or alkoxy and is on the cycloalkyl ring and the absolutestereochemistry of these ring substituents are either (R,R), (R,S),(S,R), or (S,S) and the point of attachment is the cycloalkyl ring andone of R₃ or R₄ is hydrogen.

Particularly preferred compounds of the invention are those of generalFormula I where X is —NR₃R₄ and R₃ is 2-substituted-1-indanyl and R₄ ishydrogen.

Other particularly preferred compounds of the invention are those ofgeneral Formula I where X is —NR₃R₄ and R₃ is 2-alkoxy-1-indanyl and R₄is hydrogen.

Other particularly preferred compounds of the invention are those ofgeneral Formula I where X is —NR₃R₄ and R₃ is 2(S)-alkoxy-1 (R)-indanyland R₄ is hydrogen.

Compounds provided herein can have one or more asymmetric centers orplanes, and all chiral (enantiomeric and diastereomeric) and racemicforms of the compound are included in the present invention. Manygeometric isomers of olefins, C═N double bonds, and the like can also bepresent in the compounds, and all such stable isomers are contemplatedin the present invention. Compounds of the invention are isolated ineither the racemic form, or in the optically pure form, for example, byresolution of the racemic form by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example, a chiral HPLC column, or synthesized by a asymmetricsynthesis route enabling the preparation of enantiomerically enrichedmaterial. The present invention encompasses all possible tautomers ofthe compounds represented by Formula I.

The compounds of the present invention can be synthesized using themethods outlined in Charts A–K and described below, together withsynthetic methods known in the art of synthetic organic chemistry, orvariations thereon as appreciated by those skilled in the art. Those whoare skilled in the art will recognize that the starting materials may bevaried and additional steps can be employed to produce compoundsencompassed by the invention.

Pyrimidine derivatives can be prepared as outlined in Chart A, whereinR₁ and R₂ are as defined for Formula I and X represents a halogen,preferably chloride or bromide. Compounds such as A-I can be preparedaccording to a known literature procedure [J. Org. Chem. 1983, 48,1060]. Reduction of the nitro group in A-I may be accomplished by avariety of methods known in the art, including hydrogenation withhydrogen and transition metal catalysts or the use of sodiumhydrosulfite in aqueous solutions to give A-II. Alternatively compoundssuch as A-II can be prepared by alkylation of the amino group withsuitable electrophiles (e.g., epoxides as descried in J. Med. Chem.1997, 40, 24). The aminopyridine A-II may be transformed into A-III byreductive amination using aldehydes and reducing agents such as sodiumtriacetoxyborohydride in inert solvents. The halopyridine A-III can beconverted to arylpyrimidine A-IV by a transition metal-catalyzedcoupling reaction with a metalloaryl reagent (Ar-[M]_. More commonlyemployed reagent/catalyst pairs include aryl boronic acid/palladium(0)[Stille reaction: T. N. Mitchell, Synthesis 1992, 803],arylzinc/palladium(0) and aryl Grignard/nickel(II). Palladium(0)represents a catalytic system mad of various combinations of metal/liganpairs, including, but not limited to,tetrakis(triphenylphosphine)palladium(0), palladium(II)acetate/tri(o-tolyl)phosphine,tris(dibenzylideneacetone)dipalladium(0)/tri-tert-butylphosphine anddichloro[1,1′-bis(diphenylphosphine)ferrocene]palladium(0). Nickel(II)represents a nickel-containing catalysts such as[1,2-bis(diphenylphosphino)ehtanedichloronickel(II) and[1,3-bis(dipehynlphosphino)propane]dichloronickel(II).

By changing the sequence of events in Chart A, but using the samemethods described for Chart A, compounds of formula B-IV can also beprepared as outlined in Chart B.

An alternative method for introducing the substituents R₃ and R₄ to givecompounds of formula C-IV is outlined in Chart C and may be accomplishedby a variety of methods known in the art. These methods include reactionof amine C-I with acid chlorides or anhydrides in the presence of basessuch as, but not limited to, triethylamine or pyridine in inert solventssuch as dichloromethane or toluene. The N—H group may then bedeprotonated by a strong base such as, but not limited to, alkali metalhydride, alkali metal amide, or alkali metal alkoxide in inert solventssuch as, but not limited to, tetrahydrofuran (THF), dimethylformamide(DMF) or dimethyl sulfoxide. Alkylation may be conducted using alkylhalide, suitably bromide or iodide, at temperatures ranging from 0° C.to 100° C. Reduction of the amide C-III with reducing agents such as,but not limited to, lithium aluminum hydride, borane ordiisobutylaluminum hydride in inert solvents such as, bot not limitedto, THF, ether, or toluene furnishes compounds of formula C-IV.

Another method for preparing compounds of formula D-II is illustrated inChart D. Treatment of amine D-I with base such as, but not limited to,alkali metal hyride, alkali metal amide, alkali metal alkoxide or alkalimetal carbonates in inert solvents such as, but not limited to, THF, DMFdimethyl sulfoxide or acetonitrile with or without the addition ofalkali metal iodide, followed by alkylation with alkyl halide, suitablybromide or iodide, or sulfonate at temperatures ranging from 0° C. to100° C. affords compounds of formula D-II.

Pyridine compounds of the present invention can be prepared by theroutes outlined in Charts F–K. For instance, selective metal catalyzedcross-couplings of the 2,5-dihalopyridine E-I affords 5-halo-2-pyridinesE-II (hal=halogen). The desired 3-alkoxy-6-arylpyridine E-III isobtained by heating E-II with alkoxide. Oxidation of E-III may beaccomplished with a suitable oxidant, such as m-chloroperoxybenzoic acidto afford E-IV. Heating E-IV in POCl₃ provides the halo-intermediateE-V. Conversion of E-V provides the compounds, for example,2,3-dialkoxy-6-arylpyridine E-VI by nucleophilic displacement,2-alkyl-3-alkoxy-6-arylpyridine E-VII by cross coupling and2-amino-3-alkoxy-6-arylpyridine E-VIII by amination.

3-Alkoxypyridines are also prepared by alkylation of 3-pyridinols by themethod shown in Chart F. Nitration of F-I using methods known to oneskilled in the art followed by hydroxy dediazitization provides F-II.Treating F-II with hot POCl₃ provides F-III. Cross-coupling of F-IIIwith an appropriate aryl unit affords F-IV. The nitro group of F-IV issubsequently reduced using methods known to those in the art, includingbut not limited to hydrogenation or SnCl₂, provides F-V. Hydroxydediazitization of F-V provides F-VI. Alkylation of F-VI affords thetarget 3-alkoxy-6-arylpyridine compounds F-VII.

Arylpyridines may also be prepared by construction of the pyridine ringas shown n Chart G. Condensation of malonic acids G-I with amines G-IIgives dihydroxypyridine G-III. Treatment of G-III with hot POCl₃ affordsG-IV. Selective cross coupling of G-IV to the more highly activatedhalogen proceeds to afford 2-aryl-4-chloropyridine G-V. Incorporation ofR₂ proceeds via nucleophilic substitution to provide G-VI. Hydrolysis ofG-VI affords carboxylic acid G-VII. Curtius rearrangement followed byprotection of the amine with a carbamate protecting group affordsG-VIII. Alkylation of the resultant amide provides G-IX. Deprotectionand, if desired, reductive alkylation affords the target compound G-X.

An alternative synthesis of 2-amino-3-alkoxy-6-arylpyridines isdescribed in Chart H. Iodination of H-I provides H-II, which can bealkylated to afford the corresponding 3-alkoxypyridine H-III. Bycarefully applying chemoselectivity between 2-halo and 6-iodo,halopyrazine H-III can be converted to arylpyrazine H-IV by a transitionmetal-catalyzed coupling reaction with a metalloaryl reagent (G-[M]).More commonly employed reagent/catalyst pairs include aryl boronicacid/palladium(0) (Suzuki reaction; N. Miyaura and A. Suzuki, ChemicalReview 1995, 95, 2457), aryl trialkylstannane/palladium(0) (Stillereaction; T. N. Mitchell, Synthesis 1992, 803), arylzinc/palladium(0)and aryl Grignard/nickel(II). Amination of H-IV in the presence of asuitable transition metal catalyst such as, but not limited to,palladium(II) acetate or tris(dibenzylideneacetone)dipalladium(0), aligand such as, but not limited to,1,1′-bis(diphenylphosphine)ferrocene,2,2′-bis(diphenylphosphine)-1,1′-binaphthyl,dicyclohexyl(2-biphenyl)phosphine, tricyclohexylphosphine, ortri-tert-butylphosphine, and a base such as sodium or potassiumtert-butoxide in inert solvents such as, but not limited to, toluene,ethyleneglycol dimethyl ether, diglyme, DMF, or N-methylpyrrolidinone attemperatures ranging from ambient to 100° C. provides H-V. Halogenationof H-V followed by a metal catalyzed cross-coupling reaction affords thetarget compound H-VII.

Still another route to the pyridines of interest is outlined in Chart J.Alkylation of 3-halo-5-pyridinol J-I provides J-II. Metal catalyzedcross-coupling of J-II affords J-III. Halogenation of J-III proceeds todeliver J-IV. Subsequent round of metal catalyzed cross coupling to J-IVprovides the trisubstituted pyridine J-V. A second halogenation stepgives J-VI. A final metal catalyzed cross coupling delivers the targetcompound J-VII.

Still another route to pyridines of interest is outlined in Chart K.Selective reaction of the activated bromine in the 2-position ofcommercially available K-I provides K-II. A Buchwald coupling of asuitably substituted amine provides K-III. Finally, alkylation in thepresence of a suitable base would provide K-IV.

The present invention also encompasses pharmaceutically acceptable saltsof compounds of Formula I. Examples of pharmaceutically acceptable saltsare salts prepared from inorganic acids or organic acids, such asinorganic and organic acids of basic residues such as amines, forexample, acetic, benzenesulfonic, benzoic, amphorsulfonic, citric,ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, barbaricacid, p-toluenesulfonic and the like; and alkali or organic salts ofacidic residues such as carboxylic acids, for example, alkali andalkaline earth metal salts derived from the following bases: sodiumhydride, sodium hydroxide, potassium hydroxide, calcium hydroxide,aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinchydroxide, ammonia, trimethylammonia, triethylammonia, ethylenediamine,lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, n-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like.

Pharmaceutically acceptable salts of the compounds of the invention canbe prepared by conventional chemical methods. Generally, such salts are,for example, prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17^(th) ed., MackPublishing Company, Easton, Pa., 1985, p. 1418, the disclosure of whichis hereby incorporated by reference.

In another aspect, the present invention provides a prodrug of acompound of Formula I. The prodrug is prepared with the objective(s) ofimproved chemical stability, improved patient acceptance and compliance,improved bioavailability, prolonged duration of action, improved organselectivity (including improved brain penetrance), improved formulation(e.g., increased hydrosolubility), and/or decreased side effects (e.g.,toxicity). See e.g. T. Higuchi and V. Stella, “Prodrugs as NovelDelivery Systems”, Vol. 14 of the A.C.S. Symposium Series; BioreversibleCarriers in Drug Design, ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, (1987). Prodrugs include, but are notlimited to, compounds derived from compounds of Formula I whereinhydroxy, amine or sulfhydryl groups, if present, are bonded to any groupthat, when administered to the subject, cleaves to form the freehydroxyl, amino or sulfhydryl group, respectively. Selected examplesinclude, but are not limited to, biohydrolyzable amides andbiohydrolyzable esters and biohydrolyzable carbamates, carbonates,acetate, formate and benzoate derivatives of alcohol and aminefunctional groups.

The prodrug can be readily prepared from the compounds of Formula Iusing methods known in the art. See, e.g. See Notari, R. E., “Theory andPractice of Prodrug Kinetics,” Methods in Enzymology, 112:309–323(1985); Bodor, N., “Novel Approaches in Prodrug Design,” Drugs of theFuture, 6(3):165–182 (1981); and Bundgaard, H., “Design of Prodrugs:Bioreversible-Derivatives for Various Functional Groups and ChemicalEntities,” in Design of Prodrugs (H. Bundgaard, ed.), Elsevier, N.Y.(1985); Burger's Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol.1, pp. 172–178, 949–982 (1995). For example, the compounds of Formula Ican be transformed into prodrugs by converting one or more of thehydroxy or carboxy groups into esters.

The invention also includes isotopically-labeled compounds, which areidentical to those recited in Formula I, but for the fact that one ormore atoms are replaced by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number usually found in nature.Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, iodine, and chlorine, such as ³H, ¹¹C, ¹⁴C, ¹⁸F,¹²³I, and ¹²⁵I. Compounds of Formula I that contain the aforementionedisotopes and/or other isotopes of other atoms are within the scope ofthe invention. Isotopically-labeled compounds of the present invention,for example those into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly useful in PET (positron emission tomography), and ¹²⁵Iisotopes are particularly useful in SPECT (single photon emissioncomputed tomography); all useful in brain imaging. Further, substitutionwith heavier isotopes such as deuterium, i.e., ²H, can afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements and,hence, maybe preferred in some circumstances. Isotopically labeledcompounds of Formula I of this invention can generally be prepared bycarrying out the synthetic procedures by substituting a isotopicallylabeled reagent for a non-isotopically labeled reagent.

The compounds of Formula I are antagonists at the CRF₁ receptor, capableof inhibiting the specific binding of CRF to CRF₁ receptor andantagonizing activities associated with CRF₁ receptor. The effectivenessof a compound as a CRF receptor antagonist may be determined by variousassay methods. A compound of Formula I may be assessed for activity as aCRF antagonist by one or more generally accepted assays for thispurpose, including (but not limited to) the assays disclosed by DeSouzaet al. (J. Neuroscience 7:88, 1987) and Battaglia et al. (Synapse 1:572,1987). CRF receptor affinity may be determined by binding studies thatmeasure the ability of a compound to inhibit the binding of aradiolabeled CRF (e.g., [¹²⁵I]tyrosine-CFR) to its receptor (e.g.,receptors prepared from rat cerebral cortex membranes). The radioligandbinding assay described by DeSouza et al. (supra, 1987) provides anassay for determining a compound's affinity for the CRF receptor. Suchactivity is typically calculated from the IC₅₀ as the concentration of acompound necessary to displace 50% of the radiolabeled ligand from thereceptor, and is reported as a “Ki” value. IC₅₀ and Ki values arecalculated using standard methods known in the art, such as with thenon-linear curve-fitting program GraphPad Prism (GraphPad Software, SanDiego, Calif.). A compound is considered to be active if it has an Ki ofless than about 10 micromolar (μM) for the inhibition of CRF₁ receptors.The binding affinity of the compounds of Formula I expressed as Kivalues generally ranges from about 0.5 nanomolar to about 10 micromolar.Preferred compounds of Formula I exhibit Ki value of 1 micromolar orless, more preferred compounds of Formula I exhibit Ki values of lessthan 100 nanomolar, still more preferred compounds of Formula I exhibitKi values of less than 10 nanomolar.

In addition to inhibiting CRF receptor binding, a compound's CRFreceptor antagonist activity may be established by the ability of thecompound to antagonize an activity associated with CRF. For example, CRFis known to stimulate various biochemical processes, including adenylatecyclase activity. Therefore, compounds may be evaluated as CRFantagonists by their ability to antagonize CRF-stimulated adenylatecyclase activity by, for example, measuring cAMP levels. TheCRF-stimulated adenylate cyclase activity assay described by Battagliaet al. (supra, 1987) provides an assay for determining a compound'sability to antagonize CRF activity. Alternatively, adenylate cyclaseactivity or cAMP production can be assessed in a 96/384-well formatutilizing the cAMP competitive ELISA system from Applied Biosystems(Bedford, Mass.) according to the protocols provided. Briefly, a fixedamount of diluted cAMP-alkaline phosphatase conjugate (cAMP-AP) is addedto 96 or 386-well plates containing samples from cells that werestimulated with CRF in the presence or absence of inhibitors. Anti-cAMPantibody is added to the mixture and incubated for 1 hr. Followingsuccessive wash steps, the chemiluminescent substrate/enhancer solutionis added which then produces a light signal that can be detected using amicroplate scintillation counter such as the Packard TopCount. cAMPproduced by the cells will displace the cAMP-AP conjugate from theantibody yielding a decrease of detectable signal. An example of theCRF-stimulated adenylate cyclase activity assay is provided in Example Cbelow.

Thus, in another aspect, the present invention provides a method ofantagonizing CRF₁ receptors in a warm-blooded animal, comprisingadministering to the animal a compound of the invention at amounteffective to antagonize CRF₁ receptors. The warm-blooded animal ispreferably a mammal, and more preferably a human.

In another aspect, the present invention provides a method of treating adisorder in a warm-blooded animal, which disorder manifestshypersecretion of CRF, or the treatment of which disorder can beeffected or facilitated by antagonizing CRF₁ receptors, comprisingadministering to the animal a therapeutically effective amount of acompound of the invention. The warm-blooded animal is preferably amammal, and more preferably a human.

In another aspect, the present invention provides a method for screeningfor ligands for CRF₁ receptors, which method comprises: a) carrying outa competitive binding assay with CRF₁ receptors, a compound of Formula Iwhich is labeled with a detectable label, and a candidate ligand; and b)determining the ability of said candidate ligand to displace saidlabeled compound. Assay procedure for competitive binding assay is wellknown in the art, and is exemplified in Example A.

In another aspect, the present invention provides a method for detectingCRF₁ receptors in tissue comprising: a) contacting a compound of FormulaI, which is labeled with a detectable label, with a tissue, underconditions that permit binding of the compound to the tissue; and b)detecting the labeled compound bound to the tissue. Assay procedure fordetecting receptors in tissues is well known in the art.

In another aspect, the present invention provides a method of inhibitingthe binding of CRF to CRF₁ receptors, comprising contacting a compoundof the invention with a solution comprising cells expressing the CRF₁receptor, wherein the compound is present in the solution at aconcentration sufficient to inhibit the binding of CRF to the CRF₁receptor. An example of the cell line that expresses the CRF₁ receptorand can be used in the in vitro assay is IMR₃₂ cells known in the art.

Compounds of Formula I, or a stereoisomer, a pharmaceutically acceptablesalt, or a prodrug thereof, are useful for the treatment of a disorderin a warm-blooded animal, which disorder manifests hypersecretion ofCRF, or the treatment of which disorder can be effected or facilitatedby antagonizing CRF₁ receptors. Examples of such disorders are describedherein above. They are also useful for promoting smoking cessation orpromoting hair growth.

Thus, in still another aspect, the present invention provides a methodof treating a disorder described herein above, comprising administeringto a warm-blooded animal a therapeutically effective amount of acompound of the invention. The warm-blooded animal is preferably amammal, particularly a human.

Particular disorders that can be treated by the method of the inventionpreferably include the following: anxiety-relatred disorders, such asgeneralized anxiety disorder, social anxiety disorder, anxiety withco-morbid depressive illness, obsessive-compulsive disorder, and panicdisorder, anxiety states, phobic disorders, anxiety with co-morbiddepressive illness, obsessive-compulsive disorder, post-traumatic stressdisorder, and atypical anxiety disorders; mood disorders such asdepression, including major depression, single episode depression,recurrent depression, child abuse induced depression, and postpartumdepression, bipolar disorders, post-traumatic stress disorder,dysthemia, and cyclothymia; substance abuse disorder (e.g., nicotine,cocaine, ethanol, opiates, or other drugs); inflammatory disorders suchas rheumatoid arthritis and osteoarthritis; gastrointestinal diseasessuch as irritable bowel syndrome, ulcers, Crohn's disease, spasticcolon, diarrhea, and post operative ilius and colonic hypersensitivityassociated by psychopathological disturbances or stress; and skindisorders such as acne, psoriasis, and chronic contact demertitis.

Particular disorders that can be treated by the method of the inventionmore preferably include the following: anxiety-related disorders; mooddisorders; inflammation disorders; and chronic contact demertitis.

Particular disorders that can be treated by the method of the inventioneven more preferably include anxiety-related disorders, particularlygeneralized anxiety, and mood disorders, particularly major depression.

The therapeutically effective amounts of the compounds of the inventionfor treating the diseases or disorders described above in a warm-bloodedanimal can be determined in a variety of ways known to those of ordinaryskill in the art, e.g., by administering various amounts of a particularagent to an animal afflicted with a particular condition and thendetermining the effect on the animal. Typically, therapeuticallyeffective amounts of a compound of this invention can be orallyadministered daily at a dosage of the active ingredient of 0.002 to 200mg/kg of body weight. Ordinarily, a dose of 0.01 to 10 mg/kg in divideddoses one to four times a day, or in sustained release formulation willbe effective in obtaining the desired pharmacological effect. It will beunderstood, however, that the specific dose levels for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular disease.Frequency of dosage may also vary depending on the compound used and theparticular disease treated. However, for treatment of most CNSdisorders, a dosage regimen of four-times daily or less is preferred.For the treatment of stress and depression, a dosage regimen of one ortwo-times daily is particularly preferred.

A compound of this invention can be administered to treat the abovedisorders by means that produce contact of the active agent with theagent's site of action in the body of a mammal, such as by oral,topical, dermal, parenteral, or rectal administration, or by inhalationor spray using appripropriate dosage forms. The term “parenteral” asused herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. Thecompound can be administered alone, but will generally be administeredwith a pharmaceutically acceptable carrier, diluent, or excipient.

Thus in yet another aspect, the present invention provides apharmaceutical composition comprising a compound of Formula I, astereoisomer thereof, a pharmaceutically acceptable salt thereof, or aprodrug thereof, or a pharmaceutically acceptable salt of the prodrugthereof. In one embodiment, the pharmaceutical composition furthercomprises a pharmaceutically acceptable carrier, diluent, or excipienttherefore. A “pharmaceutically acceptable carrier, diluent, orexcipient” is a medium generally accepted in the art for the delivery ofbiologically active agents to mammals, e.g., humans. Such carriers aregenerally formulated according to a number of factors well within thepurview of those of ordinary skill in the art to determine and accountfor. These include, without limitation: the type and nature of theactive agent being formulated; the subject to which the agent-containingcomposition is to be administered; the intended route of administrationof the composition; and the therapeutic indication being targeted.Pharmaceutically acceptable carriers and excipients include both aqueousand non-aqueous liquid media, as well as a variety of solid andsemi-solid dosage forms. Such carriers can include a number of differentingredients and additives in addition to the active agent, suchadditional ingredients being included in the formulation for a varietyof reasons, e.g., stabilization of the active agent, well known to thoseof ordinary skill in the art. Descriptions of suitable pharmaceuticallyacceptable carriers, and factors involved in their selection, are foundin a variety of readily available sources, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, the contents of which are incorporated herein by reference.

Compositions intended for oral use may be in the form of tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsion, hard or soft capsules, or syrups, or elixirs, andcan be prepared according to methods known to the art. Such compositionsmay contain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations.

Tablets contain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients, which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and a delay material such as glycerylmonosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending, agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexital such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, soybean oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.The oily suspensions may contain a thickening agent, for examplebeeswax, hard paraffin or cetyl alcohol. Sweetening agents such as thoseset forth above, and flavoring agents may be added to provide palatableoral preparations. These compositions may be preserved by the additionof an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occuring gums, for example gum acacia or gum tragacanth,naturally-occuring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents.

Suppositories for rectal administration of a compound of the inventioncan be prepared by mixing the compound with a suitable non-irritatingexcipient, which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Examples of such materials are cocoa butter and polyethyleneglycols.

Pharmaceutical compositions may be in the form of a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents, which have been mentioned above. Thesterile injectable solution or suspension may be formulated in anon-toxic parentally acceptable diluent or solvent, for example as asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed are water, Ringers's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid find use in the preparation ofinjectables.

Dosage forms suitable for administration generally contain from about 1mg to about 100 mg of active ingredient per unit. In thesepharmaceutical compositions, the active ingredient will ordinarily bepresent in an amount of about 0.5 to 95% by weight based on the totalweight of the composition. Examples of dosage forms for administrationof compounds of the invention includes the following: (1) Capsules. Alarge number of units capsules are prepared by filling standardtwo-piece hard gelatin capsules each with 100 mg of powdered activeingredient, 150 mg lactose, 50 mg cellulose, and 6 mg magnesiumstearate; (2) Soft Gelatin Capsules. A mixture of active ingredient in adigestible oil such as soybean, cottonseed oil, or olive oil is preparedand injected by means of a positive displacement into gelatin to formsoft gelatin capsules containing 100 mg of the active ingredient. Thecapsules were washed and dried; (3) Tablets. A large number of tabletsare prepared by conventional procedures so that the dosage unit was 100mg active ingredient, 0.2 mg of colloidal silicon dioxide, 5 mg ofmagnesium stearate, 275 mg of microcrystalline cellulose, 11 mg ofstarch, and 98.8 mg lactose. Appropriate coatings may be applied toincrease palatability or delayed adsorption.

In still another aspect, the present invention provides an article ofmanufacture comprising: a) a packaging material; b) a pharmaceuticalagent comprising a compound of the invention contained within saidpackaging material; and c) a label or package insert which indicatesthat said pharmaceutical agent can be used for treating a disorderdescribed above.

Definitions and Conventions

The following definitions are used throughout the application, unlessotherwise described.

The term “halogen” meams a group selected from —F, —Cl, —Br, —I.

The term “aryl cycloalkyl” means a bicyclic ring consisting of 9 to 14carbon atoms wherein one ring is aryl and the other ring is cycloalkylfused to the aryl ring, wherein either ring may act as a point ofattachment. The cycloalkyl ring may be fully or partially saturated inthe portion of the ring not fused to the aryl ring.

The term “substituted aryl cycloalkyl” means an aryl cycloalkyl grouphaving 1–3 substituents independently selected from halogen, —R₅, —OR₅,—S(O)_(m)R₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅, —S(O)₂NR₅R₅,—NR₅S(O)₂R₅, and —NO₂;

The term “heteroaryl cycloalkyl” means a bicyclic ring consisting of 9to 14 atoms, wherein one ring is heteroaryl and the other ring iscycloalkyl fused to the aryl ring, and wherein either ring may act as apoint of attachment. The cycloalkyl ring may be fully or partiallysaturated in the portion of the ring not fused to the aryl ring.

The term “substituted heteroaryl cycloalkyl” means a heteroarylcycloalkyl having 1–3 substituents independently selected from halogen,—R₅, —OR₅, —S(O)_(m)R₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅,—S(O)₂NR₅R₅, —NR₅S(O)₂R₅, and —NO₂;

The term “aryl heterocycloalkyl” means a bicyclic ring system containing9 to 14 atoms, wherein one ring is aryl and the other ring isheterocycloalkyl, wherein either ring may act as a point of attachment.

The term “substituted aryl heterocycloalkyl” means an arylheterocycloalkyl having 1–3 substituents independently selected fromhalogen, —R₅, —OR₅, —S(O)_(m)R₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅,—NR₅C(O)R₅, —S(O)₂NR₅R₅, —NR₅S(O)₂R₅, and —NO₂;

The term “heteroaryl heterocycloalkyl” means a bicyclic ring containing9 to 14 atoms, wherein one ring is heteroaryl and the other ring isheterocycloalkyl, wherein either ring may act as a point of attachment.

The term “substituted heteroaryl heterocycloalkyl” means a heteroarylheterocycloalkyl having 1–3 substituents independently selected fromhalogen, —R₅, —OR₅, —S(O)_(m)R₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅,—NR₅C(O)R₅, —S(O)₂NR₅R₅, —NR₅S(O)₂R₅, and —NO₂.

The term “alkyl” means both straight- and branched chain hydrocarbonmoieties having from 1–10 carbon atoms.

The term “substituted alkyl” means an alkyl moiety having 1–3substituents independently selected from halogen, —S(O)_(m)R₅, —NR₅R₅,—C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅, —S(O)₂NR₅R₅, —NR₅S(O)₂R₅, —CN,—NO₂, and Ar provided that a halogen or halogens may not be the onlysubstituent(s) on the alkyl group.

The term “haloalkyl” means is an alkyl moiety having 1 to (2v+1)independently selected halogen substituent(s) where v is the number ofcarbon atoms in the moiety.

The term “cycloalkyl” means a monocyclic or bicyclic, non-aromatichydrocarbon moiety having from 3–10 carbon atoms. A cycloalkyl mayoptionally contain 1 to 2 double bonds provided that the double bondsare not cumulated.

The term “substituted cycloalkyl” means a cycloalkyl group having 1–3substituents independently selected from halogen, —R₅, —OR₅,—S(O)_(m)R₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅, —S(O)₂NR₅R₅,—NR₅S(O)₂R₅, and —NO₂;

The term “aryl” means either phenyl or napthyl.

The term “substituted phenyl” means a phenyl group having 1–3substituents independently selected from halogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, —OR₅, SR₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅,—S(O)₂NR₅R₅, —NR₅S(O)₂R₅, and —NO₂.

The term “substituted napthyl” means a napthyl group having 1–3substituents independently selected from halogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, —OR₅, SR₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅,—S(O)₂NR₅R₅, —NR₅S(O)₂R₅, and —NO₂.

The term “heteroaryl” means a radical of a monocyclic aromatic ringcontaining five or six ring atoms consisting of carbon and 1 to 4heteroatoms each selected from the group consisting of non-peroxide O,S, and N, with appropriate bonding to satisfy valence requirements,wherein the attachment may be via a ring carbon or nitrogen atom. Theterm “heteroaryl” also includes a radical of a fused bicyclicheteroaromatic ring having about eight to ten ring atoms consisting ofcarbon and 1 to 6 heteroatoms each selected from the group consisting ofnon-peroxide O, S, and N, with appropriate bonding to satisfy valencerequirements, wherein the attachment may be via a ring carbon ornitrogen atom. Non-limiting examples of heteroaryl includes thienyl,benzothienyl, pyridyl, thiazolyl, quinolyl, pyrazinyl, pyrimidyl,imidazolyl, furanyl, benzofuranyl, benzothiazolyl, isothiazolyl,benzisothiazolyl, benzisoxazolyl, benzimidazolyl, indolyl, andbenzoxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl,pyrrolyl, isoquinolinyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pydridazinyl, triazinyl, isoindolyl, purinyl, oxadiazolyl,furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl,quinazolinyl, quinoxalinyl, naphthridinyl, and furopyridinyl.

The term “substituted heteroaryl” means a heteroaryl group having 1–3substituents independently selected from halogen, —R₅, —OR₅,—S(O)_(m)R₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅, —S(O)₂NR₅R₅,—NR₅S(O)₂R₅, and —NO₂, phenyl, substituted phenyl, napthyl, substitutednapthyl, heteroaryl, and heteroaryl derivatives.

The term “heteroaryl derivatives” means a heteroaryl group having 1–3substituents independently selected from halogen, —R₅, —OR₅,—S(O)_(m)R₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅, —S(O)₂NR₅R₅,—NR₅S(O)₂R₅, and —NO₂.

The term “heterocycloalkyl” means a 4 to 8 membered non-aromaticmonocylic ring or 6 to 12 membered non-aromatic bicyclic ring, wherein 1to 4 carbon atom(s) each is replaced with a heteromember selected fromoxygen, nitrogen, —NH—, or —S(O)_(m)— wherein m is zero, 1, or 2,wherein the ring attachment can occur at either a carbon or nitrogenatom. A heterocycloalkyl may optionally contain 1 to 3 double bonds. . .Examples of heterocycloalkyl include tetrahydrofuranyl,tetrahydropyranyl, morpholinyl, pyrrolidinyl, piperidinyl, piperazinyl,[2.2.1]-azabicyclic rings, [2.2.2]-azabicyclic rings,[3.3.1]-azabicyclic rings, quinuclidinyl, azetidinyl, azetidinonyl,oxindolyl, dihydroimidazolyl, and pyrrolidinonyl.

The term “substituted heterocycloalkyl” means a heterocycloalkyl grouphaving 1–3 substituents independently selected from halogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, —OR₅,—S(O)_(m)R₅, —NR₅R₅, —C(O)R₅, —CN, —C(O)NR₅R₅, —NR₅C(O)R₅, —S(O)₂NR₅R₅,—NR₅S(O)₂R₅, and —NO₂.

The term “pharmaceutically acceptable,” unless otherwise described,refer to those compounds, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problems orcomplications, commensurate with a reasonable benefit/risk ratio.

The term “pharmaceutically acceptable salt” refers to a salt whichretains the biological effectiveness and properties of the compounds ofthis invention and which is not biologically or otherwise undesirable.

The term “stereoisomer” refers to a compound made up of the same atomsbonded by the same bonds but having different three-dimensionalstructures which are not interchangeable. The three-dimensionalstructures are called configurations. As used herein, the term“enantiomer” refers to two stereoisomers whose molecules arenonsuperimposable mirror images of one another. The term “chiral center”refers to a carbon atom to which four different groups are attached. Asused herein, the term “diastereomers” refers to stereoisomers which arenot enantiomers. In addition, two diastereomers which have a differentconfiguration at only one chiral center are referred to herein as“epimers”. The terms “racemate” or “racemic mixture” refer to a mixtureof equal parts of enantiomers.

The term “prodrug” means compounds that are transformed in vivo to yielda compound of Formula I. The transformation may occur by variousmechanisms, such as through hydrolysis in blood.

The term “therapeutically effective amount,” “effective amount,”“therapeutic amount,” or “effective dose” is meant that amountsufficient to elicit the desired pharmacological or therapeutic effects,thus resulting in effective prevention or treatment of the disease.

The phrases “a compound of the invention,” “a compound of the presentinvention,” “compounds of the present invention,” or “a compound inaccordance with Formula I” and the like, refer to compounds of FormulaI, or stereoisomers thereof, pharmaceutically acceptable salts thereof,or prodrugs thereof, or pharmaceutically acceptable salts of a prodrugof compounds of Formula I.

The terms “treatment,” “treat,” “treating,” and the like, are meant toinclude both slowing or reversing the progression of a disorder, as wellas curing the disorder. These terms also include alleviating,ameliorating, attenuating, eliminating, or reducing one or more symptomsof a disorder or condition, even if the disorder or condition is notactually eliminated and even if progression of the disorder or conditionis not itself slowed or reversed. The term “treatment” and like termsalso include preventive (e.g., prophylactic) and palliative treatment.Prevention of the disease is manifested by a prolonging or delaying ofthe onset of the symptoms of the disease.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, practice the present invention toits fullest extent. Examples A–D are provided to illustrate biologicalassays that can be used for determining the biological properties of thecompounds of the inventions. These examples are not to be construed aslimiting the invention in scope or spirit to the specific proceduresdescribed in them Those skilled in the art will promptly recognizeappropriate variations from the procedures described in the examples.

Example A

In vitro CRF₁ Receptor Binding Assay for the Evaluation of BiologicalActivity

The following is a description of a standard in vitro binding assay forthe evaluation of biological activity of a test compound on CRF₁receptors. It is based on a modified protocol described by De Souza (DeSouza, 1987).

The binding assay utilizes brain membranes, commonly from rats. Toprepare brain membranes for binding assays, rat frontal cortex ishomogenized in 10 mL of ice cold tissue buffer (50 mM HEPES buffer pH7.0, containing 10 mM MgCl₂, 2 mM EGTA, 1 μg/mL aprotinin, 1 μg/mLleupeptin and 1 μg/mL pepstatin). The homogenate is centrifuged at48,000×g for 10 min. and the resulting pellet rehomogenized in 10 mL oftissue buffer. Following an additional centrifugation at 48,000×g for 10min., the pellet is resuspended to a protein concentration of 300 μg/mL.

Binding assays are performed in 96 well plates at a final volume of 300μL. The assays are initiated by the addition of 150 μL membranesuspension to 150 μL of assay buffer containing ¹²⁵I-ovine-CRF (finalconcentration 150 pM) and various concentrations of inhibitors. Theassay buffer is the same as described above for membrane preparationwith the addition of 0.1% ovalbumin and 0.15 mM bacitracin. Radioligandbinding is terminated after 2 hours at room temperature by filtrationthrough Packard GF/C unifilter plates (presoaked with 0.3%polyethyleneimine) using a Packard cell harvestor. Filters are washedthree times with ice cold phosphate buffered saline pH 7.0 containing0.01% Triton X-100. Filters are assessed for radioactivity in a PackardTopCount.

Alternatively, tissues and cells that naturally express CRF receptors,such as IMR-32 human neuroblastoma cells (ATCC; Hogg et al., 1996), canbe employed in binding assays analogous to those described above.

A compound is considered to be active if it has a Ki value of less thanabout 10 μM for the inhibition of CRF. Nonspecific binding is determinedin the presence of excess (10 μM) α-helical CRF.

Example B

Ex vivo CRF₁ Receptor Binding Assay for the Evaluation of BiologicalActivity

The following is a description of a typical ex vivo CRF₁ receptorbinding assay for assessing the biological activity of a test compoundon CRF₁ receptors.

Fasted, male, Harlen-bred, Sprague-Dawley rats (170–210 g) were orallydosed with test compound or vehicle, via gastric lavage between 12:30and 2:00 PM. Compounds were prepared in vehicle (usually 10% soybeanoil, 5% polysorbate 80, in dH20). Two hours after drug administration,rats were sacrificed by decapitation, frontal cortices were quicklydissected and placed on dry ice, then frozen at −80° C. until assayed;trunk blood was collected in heparinized tubes, plasma separated bycentrifugation (2500 RPM's for 20 minutes), and frozen at −20° C.

On the day of the binding assay, tissue samples were weighed and allowedto thaw in ice cold 50 mM Hepes buffer (containing 10 mM MgCl₂, 2 mMEGTA, 1 μg/mL aprotinin, 1 μg/mL leupeptin hemisulfate, and 1 μg/mLpepstatin A, 0.15 mM bacitracin, and 0.1% ovalalbumin, pH=7.0 at 23° C.)and then homogenized for 30 sec at setting 5 (Polytron by Kinematica).Homogenates were incubated (two hours, 23° C., in the dark) with [¹²⁵I]CRF (0.15 nM, NEN) in the presence of assay buffer (as described above)or DMP-904 (10 μM). The assay was terminated by filtration (PackardFilterMate, GF/C filter plates); plates were counted in Packard TopCountLSC; total and non-specific fmoles calculated from DPM's. Data areexpressed as % of vehicle controls (specific fmoles bound). Statisticalsignificance was determined using student's t-test.

Example C

Inhibition of CRF Stimulated Adenylate Cyclase Activity

Inhibition of CRF-stimulated adenylate cyclase activity can be performedas previously described [G. Battaglia et al., Synapse 1:572 (1987)].Briefly, assays are carried out at 37° C. for 10 min in 200 mL of buffercontaining 100 mM Tris-HCl (pH 7.4 at 37° C.), 10 mM MgCl₂, 0.4 mM EGTA,0.1% BSA, 1 mM isobutylmethylxanthine (IBMX), 250 units/mLphosphocreatine kinase, 5 mM creatine phosphate, 100 mM guanosine5′-triphosphate, 100 nM o-CRF, antagonist peptides (variousconcentrations) and 0.8 mg original wet weight tissue (approximately40–60 mg protein). Reactions are initiated by the addition of 1 mMATP/[³²P]ATP (approximately 2–4 mCi/tube) and terminated by the additionof 100 mL of 50 mM Tris-HCl, 45 mM ATP and 2% sodium dodecyl sulfate. Inorder to monitor the recovery of cAMP, 1 mL of [³H]cAMP (approximately40,000 dpm) is added to each tube prior to separation. The separation of[³²P]cAMP from [³²P]ATP is performed by sequential elution over Dowexand alumina columns.

Alternatively, adenylate cyclase activity can be assessed in a 96-wellformat utilizing the Adenylyl Cyclase Activation FlashPlate Assay fromNEN Life Sciences according to the protocols provided. Briefly, a fixedamount of radiolabeled cAMP is added to 96-well plates that areprecoated with anti-cyclic AMP antibody. Cells or tissues are added andstimulated in the presence or absence of inhibitors. Unlabeled cAMPproduced by the cells will displace the radiolabeled cAMP from theantibody. The bound radiolabeled cAMP produces a light signal that canbe detected using a microplate scintillation counter such as the PackardTopCount. Increasing amounts of unlabeled cAMP results in a decrease ofdetectable signal over a set incubation time (2–24 hours).

Example D

In vivo Biological Assay

The in vivo activity of a compound of the present invention can beassessed using any one of the biological assays available and acceptedwithin the art. Illustrative of these tests include the Acoustic StartleAssay, the Stair Climbing Test, and the Chronic Administration Assay.These and other models useful for the testing of compounds of thepresent invention have been outlined in C. W. Berridge and A. J. DunnBrain Research Reviews 15:71 (1990). A compound may be tested in anyspecies of rodent or small mammal.

1. A compound of Formula I,

a stereoisomer thereof, a pharmaceutically acceptable salt thereof, aprodrug thereof wherein hydroxyl, amine or sulfylhydry groups are bondedto any group that cleaves to form free hydroxyl, amino or sulfhydrylgroup respectively, or a pharmaceutically acceptable salt of a prodrugthereof, wherein: X is —NR₃R₄; G is N; R₁ and R₂ are independentlyselected from —H, —NH(alkyl), —N(alkyl)₂, —NH(substituted alkyl),—N(substituted alkyl)₂, —O(alkyl), —O(substituted alkyl), halogen,alkyl, substituted alkyl, haloalkyl, cycloalkyl, substituted cycloalkyl,aryl, substituted aryl, —CR₅R₅Ar, —OAr, —S(O)_(m)Ar, —NR₅Ar,—S(O)_(m)alkyl, —S(O)_(m)substituted alkyl, —CN, —NO₂, —OH, —NH₂, —SH,—C(O)NR₄R₅ and —C(S)NR₄R₅; R₃ and R₄ are independently selected from andaryl cycloalkyl, substituted aryl cycloalkyl, provided that when both R₃and R₄ are present one of the R₃ or R₄ is selected from a group providedherein above and the other R₃ or R₄ is selected from —H, alkyl,substituted alkyl, haloalkyl, cycloalkyl, substituted cycloalkyl, aryl,heteroaryl, heterocycloalkyl, substituted heterocycloalkyl, substitutedheteroaryl, aryl cycloalkyl, substituted aryl cycloalkyl, heteroarylcycloalkyl, substituted heteroaryl cycloalkyl, aryl heterocycloalkyl,substituted aryl heterocycloalkyl, heteroaryl heterocycloalkyl, orsubstituted heteroaryl heterocycloalkyl; Ar is selected from aryl,substituted aryl, heteroaryl, and substituted heteroaryl; and R₅ each isindependently selected from —H, alkyl, cycloalkyl, and haloalkyl,wherein alkyl may be substituted with 1–3 substituents selected fromhalogen, —O(alkyl), —NH(alkyl), —N(alkyl)₂, —C(O)NH(alkyl),—C(O)N(alkyl)₂, —NHC(O)alkyl, —N(alkyl)C(O)alkyl, and —S(O)_(m)alkyl,heterocycloalkyl, substituted heterocycloalkyl and Ar.
 2. Apharmaceutical composition comprising a compound of claim
 1. 3. Apharmaceutical composition according to claim 2 further comprising apharmaceutically acceptable carrier.
 4. A compound of claim 1 or 2wherein, in a standard in vitro CRF receptor-binding assay, the compoundexhibits a Ki value of 1 micromolar or less.
 5. A compound of claim 4wherein the compound exhibits a Ki value of 100 nanomolar or less.